SUMMARY Cancer cells arise and progress due to accumulation of genetic and epigenetic alterations that contribute to cancer phenotypes. One potential cause of these alterations are DNA sequences that are ?difficult-to-replicate? (DTR) and act as an endogenous source of replication stress. Across the genome there are two major classes of mutually overlapping DTR sequences, including mini-satellites and micro-satellites in the centromere regions and G-quadruplexes (G4s) in the telomeres. Such secondary structures formed in DTRs, if not properly resolved, may block DNA replication fork movement and lead to genome instability. However, cells have developed mechanisms to resolve these barriers for efficient and faithful DNA replication. The most well-known way to resolve G4 and other secondary structures is unwinding the structure through DNA helicases. During the last funding period, we elucidated that the nuclease/helicase DNA2 facilitates DNA replication at DTR sequences. In our preliminary studies, we showed that the DNA mismatch repair protein MSH2, a component of the MutS? complex, binds to both G4s and DNA2 and strongly stimulates DNA2 to cleave G4 structures. Radiation, elimination of DNA2 or MSH2, or lack of histone H1c ubiquitination cause G4 accumulation. Therefore, we hypothesize that: 1) DNA2 in complex with MutS? excises and repairs G4 structures to facilitate DNA replication through DTRs; 2) ubiquitinated H1c recruits the DNA2/MutS? complex onto G4-bearing DNA ends at double- strand breaks (DSBs) for homology-directed DNA repair (HDR) of DSBs; and 3) gene mutations that impair G4 resolution processes sensitize individuals to chemicals that induce or stabilize G4 structures, leading to genome rearrangements and cancer initiation. We propose to define the important molecular aspects of the G4 excision pathways during DNA replication or DSB repair. Because G4s are implicated as chromosome structural elements and epigenetic motifs that regulate gene expression, G4 excision must be tightly controlled. It is important to elucidate how the DNA2/MutS? is signaled to be recruited to the G4 structure for excision repair. We will define how H1c ubiquitination mediated by ubiquitin E3 ligases ITCH or RNF8 induces DNA2/MutS? to cleave G4 structures during DNA replication and DSB repair. Moreover, a large number of environmentally contaminating compounds (ECCs) can specifically bind to G4 structures and alter the dynamics of G4 resolution. We expect that once genetic mutations impair a G4 resolution pathway, a G4 stabilizer can act synergistically to cause DNA replication stresses, DSBs, and genome rearrangements. Therefore, we will determine if combined genetic and environmental factors that inhibit proper G4 resolution/repair show synergy in promoting genome instability and cancer initiation. Our comprehensive analyses will define the pathway for G4 excision in S phase and during DNA DSB repair via HDR, and will provide evidence that G4-stabilizing ECCs lower the cancer threshold, particularly for individuals with defects in DNA2-mediated G4 and other secondary structure resolution pathways.